ABSTRACT The broad long term objective is to improve methods of disease treatment in the liver. Hepatic fibrosis is a major cause of morbidity and mortality that affects millions of people world-wide and is usually a feature of chronic liver disease such as that caused by excessive alcohol consumption. In the United States, alcoholic liver disease (ALD) is a leading cause of GI-related deaths, with about half of the 75,000 liver disease deaths each year being related to alcohol use. A major limitation is the lack of approved therapeutics for treating either liver fibrosis or ALD. However, a new lead has emerged from our studies of exosomes that are produced by hepatocytes. Our overall objective is to establish therapeutic uses of exosomes for treating liver disease. Our Preliminary Data show that hepatocyte exosomes: (i) attenuate expression of genes that regulate fibrogenesis or activation in cultured primary hepatic stellate cells (HSC); (ii) suppress fibrogenic pathways and reverse hepatic fibrosis in vivo; (iii) attenuate damage in cultured hepatocytes exposed to ethanol or CCl4, (vi) contain relatively high levels of miR-532-5p or miR-214 that likely contribute, in part, to their therapeutic actions in fibrosis or ALD respectively especially as their respective tissue levels in the liver are individually suppressed during chronic injury; and (v) bind to target hepatocytes or HSC via cell-surface integrin ?v?3 and ?5?1. Our central hypothesis is that miR-532-5p-/miR-214 or specific integrin ligands contribute to, respectively, the therapeutic actions or cellular binding of hepatocyte exosomes. The Specific Aims to test this hypothesis are: Aim 1 - Identify miR-532-5p as a therapeutic component of hepatocyte exosomes that modulates SMAD3 in hepatocytes or HSC by using purified exosomes or cell co-culture assays, employing SMAD3 3?- UTR reporters, CTGF promoter reporters, expression of SMAD3 downstream targets, siRNA-mediated SMAD3 knockdown, and miR-532-5p over-expression or antagonism to show direct functional regulation of SMAD3 in each target cell; Aim 2 - Determine the role of hepatocyte exosomes and of exosomal miR-214 or miR- 532 in attenuating ethanol-induced liver injury by demonstrating the therapeutic effect of exosomes in ethanol diet models in vivo, on ethanol/TNF?- or LPS-mediated pathways in hepatocytes or Kupffer cells respectively, and by demonstrating that exosomal targeting of CTGF or ICAM-1 by miR-214 or of SMAD 3 by miR-532 recapitulates exosomal action in vivo and in vitro; and Aim 3 - Identify exosomal binding partners of cellular integrins by establishing the role of hepatocyte FN, VN, or CTGF in engaging integrin ?v?3 or ?5?1 on HSC or hepatocytes and thereby mediating target cell binding. The rationale is that current methods of treating liver fibrosis or ALD are inadequate and our approach is a cutting-edge and innovative solution that harnesses natural disease-suppressing properties of exosomes. The expected outcome will be a novel exosome-based therapy for treating fibrosis or alcohol-induced cell damage and altered immune function in the liver. The positive impact will be to improve the health of millions of people globally with chronic liver disease.